The present invention relates to a high purity polysaccharide containing a hydrophobic group and to a process for producing it.
In water-soluble polymeric substances, there are natural occurring polymeric substances, semisynthetic polymeric products and synthetic polymers. As the natural polymeric substances, for example, carbohydrates, such as starches and marine plant products, mucosubstances, such as gum arabic and the like, and proteins, such as glue and so on. As the semisynthetic polymeric products, there may be enumerated, for example, polymeric cellulose-like substances, such as viscose etc. As the synthetic polymers, there may be exemplified polyvinyl alcohol, polyvinylpyridine and polyglycerol. Some of the polymeric derivatives having hydrophobic groups derived from these water-soluble polymeric substances have now found their applications to medical materials, such as coating material for coating drug carriers which contain drugs. For example, it has been known that, by coating a drug carrier, such as a liposome microcapsule, microsphere, O/W emulsion or erythrocyte ghost, with a hydrophobic group-containing polysaccharide, not only the spontaneous exudation of drug from such a drug carrier is suppressed but also the cell-specific drug transference rate using such a drug carrier is improved.
In particular, those compounds in which the water-soluble polymeric substances are polysaccharides and the hydrophobic group is a steryl group, namely, polysaccharide-sterol derivatives, have already been disclosed as a polysaccharide coating material for liposomes (Japanese Patent Kokai Sho 61-69801 A), as a coating material for fat emulsion (Japanese Patent Kokai Sho 63-319046 A) and as a polymeric surfactant to be used on preparation of a polysaccharide-coated emulsion (Japanese Patent Kokai Hei 2-144140 A) and a technique for synthesizing it has been disclosed in Japanese Patent Kokai Sho 61-69801 A.
It has in recent years been widely accepted that liposome and O/W emulsion are prospective as a drug carrier. It has been reported that the chemical and physical stabilities of a drug carrier of this kind in a living body are improved by coating the drug carrier with a polysaccharide, while thereby a targettropism to a specific cell group is also revealed {Bull. Chem. Soc. Japan, 62, 791-796 (1989)}.
For synthesizing the polysaccharide-cholesterol derivative to be used therefor, a technique has hitherto been employed as described in Japanese Patent Kokai Sho 61-69801 A, which comprises the following three process steps, namely,
reacting a polysaccharide with monochloroacetic acid to synthesize a carboxymethylated polysaccharide (the first process step),
reacting the carboxymethylated polysaccharide with ethylenediamine to synthesize N-(2-aminoethyl)-carbamoylmethylated polysaccharide (the second process step) and
reacting, then, the N-(2-aminoethyl)carbamoylmethylated polysaccharide with cholesteryl chloroformate to synthesize N-{2-(cholesteryloxycarbonylamino)ethyl}-carbamoylmethylated polysaccharide (the third process step).
However, this technique disclosed in Japanese Patent Kokai Sho 61-69801 suffers from a disadvantage that the carboxyl group of the reactant in the second process step is liable to remain unreacted until the end of the process, so that the influence of the negative charge of such remaining carboxyl group on the physicochemical stability, cell-specificity, adaptability and so on of the liposome or the emulsion coated with the polysaccharide cannot be obviated. A still further problem remains in that this technique requires many process steps.
In order to resolve these problems, an alternative technique for the synthesis is proposed in Japanese Patent Kokai Hei 3-292301 A, which comprises reacting a diisocyanate with a sterol in the first step to synthesize a monoisocyanate compound having a steryl group at the xcex1-position at one end of an alkane and an isocyanato group at the xcfx89-position of the other end thereof and reacting the monoisocyanate compound with the polysaccharide in the second step to attain an easy introduction of the steryl group into the polysaccharide.
However, this technique has disadvantages in that (1) the by-product formed by the reaction of one mole of the diisocyanate compound with two moles of the sterol in the first step (in the following, referred to sometimes as the sterol dimer) is not able to be removed completely by a purification technique by dialysis or reprecipitation using ethanol and the sterol dimer will remain in the final product of polysaccharide-sterol derivative as an impurity and that (2) the unsubstituted polysaccharide which has not reacted with the monoisocyanate compound (occasionally referred to as unreacted polysaccharide) in the second step will be present in the final product of polysaccharide-sterol derivative as an impurity.
In using a polysaccharide-sterol derivative for a drug carrier or as a coating material for liposomes, a polysaccharide-sterol derivative of high purity having lower content of by-product is to be expected.
There are detailed reports as to the above-mentioned polysaccharide-sterol derivatives and as to amphiphilic composite products in which a hydrophobic group other than sterol is bound to a water-soluble polymeric substance other than polysaccharide, such as alkyl diesters of polyethylene glycol {Dojin News No. 85, p 3-11 (1997)}.
However, there is no report up to date as to a hydrophobic group-containing water-soluble polymeric substance of high purity, due to existing difficulty in the production of such substance, as mentioned above.
The first object of the present invention is to propose a process permitting production of a high purity polysaccharide containing a hydrophobic group with a low content of impurities, such as an unsubstituted polysaccharide and sterol dimer, in an efficient and easy manner.
The second object of the present invention is to provide a high purity polysaccharide containing a hydrophobic group obtained by the production process mentioned above.
The inventors had performed sound researches in respect of the problems in the prior art given above and reached the discovery that a high purity polysaccharide containing a hydrophobic group was able to be obtained by employing a combination of technical measures of use of a solvent based on a ketone in the reprecipitation process and of purification by means of an ultracentrifugation or of purification with an aprotic polar solvent, whereby the present invention has been completed. Thus, the present invention consists in the high purity polysaccharide containing a hydrophobic group and in the process for producing it as given below:
(1) A process for producing a high purity polysaccharide containing a hydrophobic group, comprising
a first process step of producing an isocyanate group-containing hydrophobic compound, wherein one mole of a hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or of a sterol is reacted with a diisocyanate represented by OCNxe2x80x94R1NCO in which R1 is a hydrocarbyl of 1-50 carbon atoms and
a second process step of producing the polysaccharide containing hydrophobic group composed of the hydrocarbon group of 12-50 carbon atoms or of the steryl group, wherein the isocyanate group-containing hydrophobic compound obtained in the first process step is reacted with one or more polysaccharides,
wherein the reaction product in the second process step is purified using a solvent based on a ketone.
(2) The process as defined in the above (1), wherein the polysaccharide is selected from the group consisting of pullulan, amylopectin, amylose, dextran, hydroxyethyl cellulose, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan and water-soluble cellulose.
(3) The process as defined in the above (1) or (2), wherein the solvent based on a ketone comprises one or more selected from the group consisting of acetone, methyl ethyl ketone, diethyl ketone and diisopropyl ketone.
(4) The process as defined in any one of the above (1) to (3), wherein the hydrophobic group-containing polysaccharide has a group represented by xe2x80x94XH in which X is an oxygen atom or a nitrogen-containing group represented by NY with Y being a hydrogen atom or a hydrocarbyl of 1-10 carbon atoms wherein 0.1-10 xe2x80x94XH groups per 100 monosaccharide units constituting the polysaccharide are replaced by one or more hydrophobic groups represented by the formula (1), namely, 
in which X is the same as given above, R1 denotes a hydrocarbyl having 1-50 carbon atoms and R2 denotes a hydrocarbon group of 12-50 carbon atoms or a steryl group.
(5) The process as defined in the above (4), wherein R2 in the formula (1) denotes a steryl group.
(6) The process as defined in any one of the above (1) to (5), wherein the content of the hydrophobic group-containing polysaccharide in the product purified using the solvent based on a ketone is as high as 80% by weight or more.
(7) The process as defined in the above (6), wherein the content of unsubstituted polysaccharide is as low as 20% by weight or less.
(8) The process as defined in the above (6) or (7), wherein the product has a content of the impurity product, in which both the two NCO groups in the diisocyanate are reacted with the hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or with the sterol, as low as 0.05% by weight or less.
(9) The process as defined in any one of the above (1) to (8), wherein the product purified using a solvent based on ketone is subjected to a further purification by dispersing the product finely in water under an ultrasonic treatment, with subsequent ultracentrifugal separation.
(10) The process as defined in the above (9), wherein the content of the hydrophobic group-containing polysaccharide in the purified product from the ultracentrifugal separation is as high as 98% by weight or more.
(11) The process as defined in the above (10), wherein the content of unsubstituted polysaccharide is as low as 2% by weight or less.
(12) The process as defined in the above (10) or (11), wherein the content of the impurity product, in which both the two NCO groups in the diisocyanate are reacted with the hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or with the sterol, is as low as 0.05% by weight or less.
(13) The process as defined in any one of the above (1) to (8), wherein the product purified using the solvent based on a ketone is further subjected to a purification procedures comprising dissolving the product in an aprotic polar solvent, admixing water to the resulting solution to cause the unsubstituted polysaccharide to be transferred to the aqueous phase and removing the aqueous phase separated by phase separation.
(14) The process as defined in the above (13), wherein the further purification of the product purified using the solvent based on a ketone is performed by dissolving the product in the aprotic polar solvent of an amount of 3-50 times the weight of the product and admixing water with the resulting solution in an amount of at least 5 times the weight of the solution.
(15) The process as defined in the above (13) or (14), wherein the aprotic polar solvent comprises one or more selected from the group consisting of N,N-dimethyl-formamide, N,N-dimethylacetamide and dimethyl sulfoxide.
(16) The process as defined in any one of the above (13) to (15), wherein the content of the hydrophobic group-containing polysaccharide in the product purified using the aprotic polar solvent is as high as 98% by weight or more.
(17) The process as defined in the above (16), wherein the content of the unsubstituted polysaccharide is as low as 2% by weight or less.
(18) The process as defined in the above (16) or (17), wherein the content of the impurity product, in which both the two NCO groups in the diisocyanate are reacted with the hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or with the sterol, is as low as 0.02% by weight or less.
(19) A high purity product of a polysaccharide containing a hydrophobic group which contains at least 80% by weight of the polysaccharide containing a hydrophobic group, wherein the polysaccharide is one having a group represented by xe2x80x94XH in which X is an oxygen atom or a nitrogen-containing group represented by NY with Y being a hydrogen atom or a hydrocarbyl of 1-10 carbon atoms wherein 0.1-10 xe2x80x94XH groups per 100 monosaccharide units constituting the polysaccharide are replaced by one or more hydrophobic groups represented by the formula (1), namely, 
in which X is the same as given above, R1 denotes a hydrocarbyl having 1-50 carbon atoms and R2 denotes a hydrocarbon group of 12-50 carbon atoms or a steryl group,
said polysaccharide containing a hydrophobic group being obtained by a process comprising
a first process step of producing an isocyanate group-containing hydrophobic compound, wherein one mole of a hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or of a sterol is reacted with a diisocyanate represented by OCNxe2x80x94R1xe2x80x94NCO in which R1 is a hydrocarbyl of 1-50 carbon atoms,
a second process step of producing the polysaccharide containing hydrophobic group composed of the hydrocarbon group of 12-50 carbon atoms or of the steryl group, wherein the isocyanate group-containing hydrophobic compound obtained in the first process step is reacted with one or more polysaccharide, and
purifying the reaction product from the second process step using a solvent based on a ketone.
(20) The high purity product of a polysaccharide containing a hydrophobic group as defined in the above (19), wherein the polysaccharide is selected from the group consisting of pullulan, amylopectin, amyliose, dextran, hydroxyethyl cellulose, hydroxyethyl dextran, mannan, levan, inulin, chitin, chitosan, xyloglucan and water-soluble cellulose.
(21) The high purity product of a polysaccharide containing a hydrophobic group as defined in the above (19) or (20), wherein R 2 in the formula (1) is steryl.
(22) The high purity product of a polysaccharide containing a hydrophobic group as defined in any one of the above (19) to (21), wherein the content of unsubstituted polysaccharide is as low as 20% by weight or less.
(23) The high purity product of polysaccharide containing a hydrophobic group as defined in any one of the above (19) to (22), wherein the content of the impurity product, in which both the two NCO groups in the diisocyanate are reacted with the hydroxyl group-containing hydrocarbon having 12-50 carbon atoms or with the sterol, is as low as 0.05% by weight or less.
(24) The high purity product of a polysaccharide containing a hydrophobic group as defined in any one of the above (19) to (23), wherein the product purified using the solvent based on a ketone is subjected to a further purification by dispersing the product finely in water under an ultrasonic treatment, with subsequent ultracentrifugal separation.
(25) The high purity product of a polysaccharide containing a hydrophobic group as defined in any one of the above (19) to (23), obtained by subjecting the product purified using the solvent based on a ketone to a further purification procedures comprising dissolving the product in an aprotic polar solvent, admixing water with the resulting solution to cause the unsubstituted polysaccharide to be transferred to the aqueous phase and removing the aqueous phase separated by phase separation.